Peripheral arterial disease (PAD) is estimated to affect as many as 20% American adults over 65 years of age and results decreased lower extremity function and overall quality of life. Therapeutic angiogenesis, stimulation of new blood vessels to replace the function of dysfunctional, diseased arteries, represents a promising, relatively new approach that could restore peripheral circulation and quality of life for PAD patients. The mouse hind limb ischemia model is a commonly used preclinical system for testing new therapeutic angiogenesis strategies including growth factor delivery, gene therapy, and cell therapy. The hind limb ischemia model also provides a well-defined and previously-characterized platform for studying the biological mechanisms of neovascularization in vivo. However, analytical techniques for characterization of the angiogenic response in this model system are at present lacking due to one or more shortcomings including lack of quantitative data provided, user subjectivity, inadequate spatial resolution, lack of longitudinal in vivo imaging capability, and the requirement for procuring multiple sets of mice for analysis of both vessel structure and function. The central goal of the current proposal is to develop an optical, intravital imaging technique that can be used to acquire co-registered, 3D image data on vessel morphology, blood flow, and oxygenation. The acquired quantitative data sets will provide a significant improvement in the amount of complementary structure-function data that can be taken from a single mouse under a single dose of anesthesia. Furthermore, throughput of preclinical and basic studies should be accelerated and experimental costs decreased because repeated imaging can be carried out in a single mouse. Two aims are proposed that span (1) technique development and (2) technique validation relative to traditional gold standard techniques. The proposed technique has the potential to provide innovative, new tools of high impact to angiogenesis researchers and to accelerate development of new therapeutic strategies for treatment of significant clinical problem of PAD. PUBLIC HEALTH RELEVANCE: Small animal models of peripheral limb ischemia play an integral role in understanding the basic mechanisms of neovascularization of ischemic tissues and preclinical testing of pro-angiogenic therapies. However, a significant shortcoming exists in the availability of quantitative, high resolution methodologies for longitudinal assessment of blood vessel morphology and function within living animals. Here, we propose to develop a novel optical imaging approach capable of simultaneous assessment of vascular morphology, blood flow, and oxygenation in living mice without the need for exogenous contrast agent administration. This technique will enable more robust assessments of vascular structure/function and is anticipated to accelerate new discoveries on the mechanistic control of angiogenesis and the development of therapeutic revascularization technologies.